The TopoisomeraseII (TopII) family is an important class of topoisomerases that produces transient 5? DNA-protein crosslinks [1, 2]. Drugs targeting these enzymes act by stabilizing the crosslinks, producing a covalent DNA-protein complex [3, 4]. Despite their clinical efficacy, drugs that target TopII (TopII poisons) may lead to negative side-effects, including secondary malignancies. The repair of TopII-DNA complexes is poorly understood. Recently, a human 5?- tyrosine phosphodiesterase (hTDP2) has been identified for the excision of TopII-DNA adducts [5, 6]. As hTDP2 repairs the cytotoxic adducts produced by TopII poisons, it is likely that effective inhibition of hTDP2 can be targeted to improve the efficacy of TopII poisons and reduce the side-effects. It has been shown that the knockdown/knockout of TDP2 activity in A549 (non-small cell lung cancer cell line, NSCLC) and DT40 cells increased sensitivity to the TopII targeting agent etoposide, demonstrating that hTDP2 can be a target for adjuvant chemotherapy [7]. Our hypothesis is effective inhibition of hTDP2 will increase the cancer cell killing efficacy of TopII poisons like etoposide [8]. Therefore, hTDP2 inhibitor screening, especially in a high-throughput manner, will expedite drug discovery.

Assay Overview:The purpose of this assay is to confirm tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitory activity of compounds identified as active in a set of previous experiments entitled, "Epi Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)" (AID 720702). TDP2 is a divalent cation-dependent enzyme that repairs TopII-associated DNA strand breaks. It is hypothesized that inhibitors of TDP2 may serve as useful adjuvants in combination with cancer drugs such as etoposide.In this biochemical assay, recombinant human TDP2 protein is incubated at 37 degrees Celsius with the T5PNP substrate in the presence of Mg2+-containing assay buffer.T5PNP is a substrate for snake venom phosphodiesterase, as well as a substrate for TDP2. As a substrate for TDP2, T5PNP is used to mimic the TopII-DNA complex. TDP2 cleaves the phosphodiester bond in T5PNP, and the chromogenic p-nitrophenol group is released. As time increases the TDP2 enzyme will increasingly catalyze hydrolysis of the T5PNP substrate, resulting in increased release of p-nitrophenol and detection at 415nM wavelength. Compounds are tested in triplicate at a final nominal concentration of 12.8 uM.Protocol Summary:Prior to the start of the assay, 2 ul of a solution containing T5PNP subtrate (final concentration 5mM) in assay buffer (50mM Tris-HCl pH7.5, 1mM DTT, 1mM MgCl2, 50mM KCl and 100ug/ml BSA) was dispended into a all wells of a 1536 well plate. Next, 39nL of test compound in DMSO or DMSO alone (1% final concentration) was added to the appropriate wells. The assay was started by dispensing 1 ul of a solution contanting TDP2 enzyme (120nM final concentration) in assay buffer to wells in columns 4-48 and 1ul of assay buffer alone to wells in columnes 1-3. Plates were centrifuged and incubated for 2hrs at 37 degrees Celsius at which time fluorescence intensity was measured (Ex. 405nm and Em. 405nm) using a EnVision microplate reader (Perkin Elmer).Prior to further calculations, the following formula was used to calculate Epi Absorbance (EPIABS)EPIABS = -log10( sample / background)Where:Sample is defined as the fluorescent intensity of wells containing test compounds or DMSO.Background is defined as the fluorescent intensity of wells containing buffer and T5PNP only.The % inhibition for each well was then calculated as follows:%_Inhibition = ( EPIABS_Test_Compound - MedianEPIABS_Low_Control ) / ( MedianEPIABS_High_Control - MedianEPIABS_Low_Control ) * 100Where:Low_Control is defined as wells containing DMSO, TDP2 and T5PNP.Test_Compound is defined as wells containing test compound, TDP2 and T5PNP.High_Control is defined as wells containing only Buffer and T5PNP.PubChem Activity Outcome and Score:The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 720702) was declared active.The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.The PubChem Activity Score range for active compounds is 100-25, and for inactive compounds 25-0.List of Reagents:TDP2 (supplied by Assay Provider)Tris Base(Sigma, 93349)DTT (Sigma, 43815)BSA (Sigma, A2153)MgCl2 (Sigma,M2670)KCl (Sigma, P9333)T5PNP (Sigma, T4510)1536 well plates (Corning 7254)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.